The main criterion for inclusion to the studies was the detection of neurological symptoms resembling neuroborreliosis (meningitis, neck stiffness, facial nerve palsy and cerebrovascular diseases).
All patients came from central Poland. They all had tick bite history. All patients included were diagnosed with neuroborreliosis. These patients were not part of the previous study. We don't have information about erythema migrans history in the population patients. Samples were taken from patients within 1-2 days of the onset of neurological symptoms. All patients had antibiotic therapy (samples were taken in 80% of patients before antibiotic therapy, 20% after treatment initiation) Blood serum and cerebrospinal fluid (CSF) samples from 133 (72 women and 61 men) patients with clinical suspicion of neuroborreliosis were investigated. All patients agreed to the use of their material for scientific research. A total of 266 samples (two samples per patient: one serum and one CSF) were collected between 2010 and 2018 at the Laboratory of Rickettsiae, Chlamydiae and Spirochetes, NIPH-NIH. Diagnosis was established by detection of IgM and/or IgG specific antibodies to Borrelia burgdorferi with ELISA (DRG MedTec, Germany) in sera and/or CSF. The specificity of positive ELISA results in sera was confirmed with the Western-blot test (DRG MedTec, Germany).
As positive results in: Western-blot IgM class antibodies was accepted the presence of specific antibodies reacting with at least two of the following fractions: p18; p39, OspC, p41 and VlsE or strong reaction with the OspC antigen; Western-blot IgG class antibodies was accepted the presence of specific antibodies reacting with at least two of the following fractions: p83, p58, p41(int.), p39, OspC, p18 i VlsE.
The status of all Borrelia-positive samples was confirmed by nested PCR for the glycerophosphodiester phosphodiesterase (glpQ) B. miyamotoi gene using the protocol described by Fomenko et al. , with modifications. Bacterial DNA from the collected materials (266 samples; 133 samples of CSF and 133 blood samples) was extracted with a SYNGEN Tissue DNA kit (SYNGEN BIOTECH).
Nested PCR reactions were performed with Gold Hot Start PCR MIX LOAD (Syngen Biotech, Poland) using forward and reverse primers under the following conditions: 15 min at 95°C, then 35 cycles of 30 s 95°C, 60 s 59°C, 30 s 72°C and finishing with 10 min at 72°C for external primer pair (Q1: 5’-CACCATTGATCATAGCTCACAG-3’ and Q2: 5’-CTGTTGGTGCTTCATTCCAGTC-3’) and internal primer pair (Q3: 5’-GCTAGTGGGTATCTTCCAGAAC-3’ and Q4: 5’-CTTGTTGTTTATGCCAGAAGGGT-3’): 15 min at 95°C; 35 cycles of 30 s 95°C, 60 s 52°C, 30 s 72°C and finishing with 10 min at 72°C. Each run of the PCR test included positive (B. miyamotoi DNA obtained during annual participation in proficiency tests, INSTAND Germany, DNA concentration 5×104 organisms/mL) and negative controls (H2O). Amplified amplicons were analyzed using electrophoresis with 2% agarose gel (Basica LE, Prona) stained with Midori Green Advance DNA Stain (Nippon Genetics Europe GmbH). The nested PCR products (425 bp fragments) were sequenced and identified using BLAST software by comparison with sequences available in GenBank.
The nested-PCR-positive sample was additionally analyzed with primers targeting a 723 bp fragment of glpQ gene. The PCR was performed with Gold Hot Start PCR MIX LOAD (Syngen Biotech, Poland) using forward 5’-ATGGGTTCAAACAAAAAGTCACC-3’ and reverse primers 5’-CCAGGGTCCAATTCCATCAGAATATTGTGCAAC-3’ under the following conditions: 15 min 95°C, then 40 cycles of 30 s 95°C, 30 s 65°C, 90 s 72°C and finishing with 10 min at 72°C (protocol described by Wagemakers et al. 2017, with modifications) . The melting temperatures of the used primers varied by 15°C PCR with gradient temperature (from 53°C to 68°C) was performed and the most appropriate temperature (65°C) was chosen. Amplicons were sequenced and identified using BLAST software by comparison with sequences available in GenBank.
B. burgdorferi DNA was searched in CSF samples for rule out coinfection case.
The PCR was performed also with Gold Hot Start PCR MIX LOAD (Syngen Biotech, Poland) using forward 5’-ATGGGTTCAAACAAAAAGTCACC-3’and reverse primers 5’- OA 149 (sequence 5'-TTA TGA AAA AAT ATT TAT TGG GAAT-3 ') and OA 319 (sequence 5' CTT TAA GCT CAA GCT TGT CTA CTGT-3 ') complementary to the fragment of the OspA gene for B. burgdorferi (product 170bp) under the following conditions: 15 min 95°C, then 40 cycles of 60 s 95°C,60 s 55°C, 60 s 72°C and finishing with 3 min at 72°C .